The basic leucine zipper transcription factor, C/EBPbeta, is critical for growth and differentiation of the mammary gland. Epithelial cell proliferation in early pregnancy and differentiation at late pregnancy are severely impaired in C/EBPbeta null mice, which fail to lactate. Alternative translation of the intronless C/EBPbeta gene produces three different protein isoforms. C/EBPbeta-1 and -2 are transactivators, whereas C/EBPbeta-3 lacks a transactivation domain and inhibits transcription. Although differences between C/EBPbeta-1 and -2 have received little attention, our studies show that they are functionally distinct. Ectopic C/EBPbeta-2 expression transforms mammary epithelial cells (MECs) in vitro. The cells become anchorage independent, undergo an epithelial to mesenchymal transition (EMT), and gain invasive growth characteristics. In contrast, C/EBPbeta-1 expression blocks the invasive growth of metastatic breast cancer cell lines in culture. In this application we propose to examine the mechanistic basis underlying the functional dichotomy in C/EBPbeta-1 vs. -2 expression. These two transactivators differ by only 21 N-terminal amino acids present in C/EBPbeta-1, but absent from C/EBPbeta-2. We hypothesize that C/EBPbeta-1 and -2 undergo isoform-specific posttranslational modifications essential for their different functions. In support of this hypothesis, we have recently shown that C/EBPbeta-1, but not C/EBPbeta-2, is conjugated to the small ubiquitin-like modifier proteins, SUMO2 and SUMO3. We will determine whether SUMO-conjugation is necessary for the inhibition of invasive growth by C/EBPbeta-1. We will also investigate whether SUMO2,3 modification is required for protein-protein interactions with the Swi/Snf chromatin remodeling complex and/or affects the localization of C/EBPbeta-1 is subnuclear speckles. In contrast, C/EBPbeta-2 is targeted by multiple protein kinases, some of which are downstream of Ras activation. Indeed, C/EBPbeta has been shown to be an essential target of oncogenic Ras signaling in skin tumorigenesis and NIH 3T3 transformation. We will examine the requirement for C/EBPbeta-2 phosphorylation by ERKI/2 and p90Rsk-2 in mammary epithelial cell transformation. In human breast cancer, Ras-dependent signaling pathways are often activated by alterations in receptor tyrosine kinases. Therefore, we plan to determine whether receptor tyrosine kinase activation will synergize with C/EBPbeta-2 in transforming mammary epithelial cells. Because C/EBPbeta-2 promotes, whereas C/EBPbeta-1 inhibits, the invasive growth of MECs, misregulated expression of these two transactivator isoforms could contribute to growth and metastasis in breast cancer. Interestingly, C/EBPbeta-2 is undetectable in normal human breast tissue (obtained from reduction mammoplasty) where only C/EBPbeta-1 is expressed. However, 60% of primary breast tumors examined showed a high level of C/EBPbeta-2 expression, and moderate expression was detected in another 30% of the samples. Moreover, all breast cancer cell lines in culture express C/EBPbeta-2, but none express C/EBPbeta-1. We have recently generated mice carrying an MMTV-driven C/EBPbeta-2 transgene, and significantly, virgin females exhibit precocious, hyperplastic mammary gland development. We will continue to study these animals to determine if females with hyperplasia go on to develop neoplasia and/or have accelerated development of metastatic carcinoma when crossed with other mouse models of breast cancer. Once cancer cells have metastasized, breast cancers are largely incurable even with state-of-the-art approaches. These studies will provide important insights into how misregulated C/EBPbeta isoform expression may contribute to the development of metastatic mammary carcinoma.